RICERCA ITALIANA     

Biocompatible soft matter systems made of surfactants and macromolecules

Università degli Studi di Roma "La Sapienza"

Abstract

The present program is focused on the investigation of “soft matter systems”, namely water-based self-assembling systems, either sol or gel, made of biocompatible surfactants and macromolecules. Here from, the term macromolecule defines a variety of chemical species, including functionalised polysaccharides, proteins and nucleic acids.
In the program there is a balance between fundamental and applied aspects. The former purposes are directed to getting a deeper insight into structural and other physico-chemical properties, like the transport mechanisms and the kinds of interaction, profitable in order to optimise final formulations characteristics. This will be accomplished by means of the synergetic interpretation of results obtained through various up to date techniques, which encompass a huge range of very different time- and length- scales.
The outcomes are expected to lead to the optimisation of the formulation of systems (micellar aggregates, vesicles, gel, vesicular gels, liquid crystalline or bicontinuous phases) with tailored properties as to dispersion, adsorption, stabilisation and rheology, important for controlled release and cosmetic. To assess the functional synergy associated to the macromolecule-surfactant (P-T) interaction, simple systems, i.e. binary aqueous systems concerning just one macromolecule or surfactant, or at most ternary mixtures, will be studied.
The investigations are embedded in the logic of “eco-sustainable development” since it will make use of natural products and of surfactants from biocompatible synthesis, with the least environmental impact.
The macromolecules considered i.e. chitosan, ionic derivatives of cellulose, albumine, lysozyme and DNA, are of natural origin and are representative members of classes of compounds, or possess selected functionalities. The biodegradability and biocompatibility of the surfactants employed is a pressing need for friendly formulations. The amino acid-based surfactants hydrophilic (anionic, cationic, nonionic) and hydrophobic (mono or dimeric alkyl chains) moieties can be synthesised using renewable raw materials. They show low toxicity, antimicrobial activity and quick biodegradation. Moreover, monoolein and sodium oleate are natural molecules like mono, di and trihydroxylated bile acid salts, endogenous surfactants that present peculiar chemical structures, self-assembling and interaction properties, different to those of the classical surfactants.
The investigations in the sol phase will concern:
- Equilibrium properties, through surface tension, volumetry, calorimetry (ITC) colligative properties, fluorescence, EMF and circular dichroism. These will afford critical association parameters, interactions and their dependence on surfactant, macromolecule as well as on composition.
- Transport properties, by determining mutual and self-diffusion coefficients, dynamic light (DLS) and X-ray diffusion (XRD), or zeta potential. Information on the mobility of molecules and P-T adducts (complexes) shall be obtained.
- Scattering: SANS, SAXS and SLS (static light scattering) will provide aggregate or molecular sizes, structure and polydispersity
- Spectroscopies: multinuclear NMR, EPR, Raman, IR and dielectric relaxation spectroscopy will provide information on the nature of binding sites, their microenvironment and dynamics.
As to gels and liquid crystals, studies shall deal with:
- Polymorphic behaviour and optimal conditions for gelling;
- Characterisation by PGSE-NMR, EPR, SAXS, SANS, slow-flow (fast-flow) rheology, rheo-NMR, optical and SEM microscopy, NMR relaxation and optical methods;
- Optimisation of controlled release conditions.
More specifically, these studies are aimed at clarifying aspects related to the species self assembly, their segregation in specific domains, and functional aspects associated with the P-T interactions. <<<

Principal Investigator

Nicolae Viorel Pavel Università degli Studi di ROMA "La Sapienza"

Research Objectives

The purpose of this research program deals with the study of supramolecular aqueous systems based on biocompatible surfactants and biocompatible macromolecules both in sol and gel phases.
The study, carried on by five research units (RU) is balanced between the formulation of systems having particular dispersing, stabilising, rheological, and controlled release properties, and their characterization at the fundamental physico-chemical level. In particular, the aim is to deepen the structural study and the interaction and transport mechanisms, required for optimising the properties of formulations. It is our intention to unify the results obtained with different techniques operating on different length and time scales and to correlate them with the rheological properties.

The project is devoted to the study of different complexity systems:
a) Aggregate surfactants in aqueous solution [micelles (M), vesicles (VES)];
b) Sol systems containing water, polymer and surfactant (M or VES);
c) Liquid crystalline or bicontinuous phases (cubosome, hexasome);
d) Polymeric gels of water and polymer;
e) Gel systems made by water, polymer and surfactant (M or VES);
f) Gels in the presence of actives: detailed exam and rationalization of solubilising and transport properties;
g) Surfactant- protein, or DNA, systems in sol and gel phases

The systems under consideration will require a parallel work pointing to:
i)synthesis of biocompatible surfactants and their modellization aiming at improving the properties of the systems.
ii)Development and implementation of experimental characterization techniques, proper for the specific systems.
iii)Development of computational techniques able to correlate the structural with macroscopic properties.

In order to define specific tasks, the project has been divided into the following workpackages, each of which coupled with the participating UR:
[SOL] Self-aggregated systems in the liquid phase (RM, CS, PI, NA, TS)
[HGE] Hydrogels (NA, CS)
[VGE] Vesicular gels (RM, CS)
[MES] Liquid-crystalline structures (CS, RM,TS)
[BIO] Biological structures (RM, PI, NA, TS)
[SYN] Synthesis of new biocompatible surfactants and of biocompatible modified polymers (CS, a Spanish group, NA, PI)
[MET] development and implementation of experimental techniques of characterization, appropriate for the studied systems (RM, CS, NA, TS)

The UR are the following: UR1-Roma (RM), UR2-Cosenza (CS), UR3-Pisa (PI), UR4-Napoli (NA) and UR5-Trieste (TS).


MOTIVATION
One of the main goals of present project is to formulate systems, belonging to the soft matter field, such as micellar aggregates, vesicles, gels, vesicular gels, liquid crystals, characterized by good biocompatibility and good protective and releasing properties.

All the activities fall within the logic of the “sustainable growth” as only natural products and synthetic biocompatible surfactants will be used, in view of their lower environmental impact.

Recent development of drugs based on proteins and peptides largely increased the number of systems suitable for their transport and controlled release. The usefulness of release systems is double: i) protection of drugs from a possible drastic metabolism of human body that would thwart their action, ii) possibility of keeping the drug concentration constant, or almost constant, in time avoiding the effect of peak concentrations. Problems of protection and released control are also pertinent to the case of topical administrations.

Gels are complex systems having an increasing use in the controlled drug release. Gels have at the same time the elastic properties of solids and the viscosity of liquids. Aqueous P-T mixtures make gels (called hydrogels) that can trap hydrophilic and/or hydrophobic molecules. The complex structures present inside gels and the host-guest interactions involved are responsible of a low diffusivity of guest molecules causing their slow release.

Engineering of VES is developing continuously thanks to their extended applications, from the biomedical to the food and the nanoscience field. The VES structures are made by one ore more double-layers, formed by the self-association of surfactants, lipids, or block copolymers. Like membranes, they create a separate compartment inside which they can trap molecules or macromolecules. For this reason VES are employed as nano-reactors or as delivery vehicles of drugs, DNA and targets for various non invasive biomedical research techniques.

Complex systems of VES and polymers may exhibit peculiar properties: mechanical properties due to the polymer gels, and trapping and releasing properties due to the VES. Engineering of these systems is still at an empirical level. Interactions are ruled by hydrophobic and electrostatic forces, whose effects are still out of control: in some cases they can lead to the formation of vesicular gels, with VES immobilized and made stable by the presence of polymer chains.

Problems concerning gel systems made up by polymers and surfactants (M or VES) are strictly related with the study of the interactions between the same components in the sol phase.
The DNA binding on VES and its inclusion into them is the research subject of various groups in the world. Besides the use of VES obtained from lipids and their mixtures, sometimes unstable, catanionic VES obtained by mixing surfactants of opposite charges appear very promising. The studies put in evidence the DNA binding and packing on VES and give some indication on possible strategies for transfection. Its adsorption on the VES surface controls the transfer of the biopolymer inside the VES, influencing both DNA transfection and drugs vehiculation. Surface adsorption and charge modification in such systems must be studied carefully with adequate techniques.
The effects of a surfactant on a protein depends on its nature. The surfactant characteristics establish its capability to bind, unfold, denaturate, or stabilize the protein. "Hard" surfactants bind proteins strongly disrupting their native structure and making them inactive.

Some natural ionic and nonionic surfactants, that interact weakly with protein molecules, are considered "mild" and do not promote protein precipitation.
Interaction models in diluted and concentrated systems of classical detergents or polymers (like PEO) with proteins like albumins, lysozime, myoglobin etc., have been proposed for their structural and thermodynamic characterization. Various surfactant-protein models are present in the literature: necklace, decorated micelles, flexible coil, able to interpret different P-T systems.
Diseases due to condensation (cataract, rheumatic diseases) are pathologies starting with a solubility loss of some substances thus producing a condensed phase. Very often the condensing molecules are proteins, so that agents contrasting denaturation are promising drugs for the treatment of these diseases. There is evidence that endogenous surfactants as the bile salts have such an antidenaturant effect. <<<

Timescale

24 months

National and international background

Systems raising from surfactant and surfactant-macromolecule aggregation belong to soft matter field. They are presently of great interest from both an academic and applicative viewpoint. These systems, presenting a variety of structural and chemical characteristics, are well suited in the building up of materials such as microemulsions, vescicles, gels, liquid crystals, bicontinuos phases. They can also give rise to tridimensional networks organized at different levels: from the molecular to nanometer to millimeter scales.

During the last years, P-T systems found numerous applications in industrial, alimentary, environmental, medical fields, thus raising the scientific interest for the study of their aggregation ability, behavior and structure. Actually literature on this subject is very wide, although we are bound to mention only few papers [1-12] because of the shortness of this document.

In summary, the goal of this project is:
1)Formulation and characterization of new vescicular systems, lyotropic liquid crystals and bicontinuos phases.
2)Formulation of bio-compatible gels made of a polymeric network including surfactants as monomers, micelles or VES
3)Selection of gels for drug delivery presenting good solubilizing and protective ability, rheological and diffusive properties.
4)Study, at micro and macro levels, of P-T and DNA-VES interactions
We will focus on bio-compatible and bio-degradable amphiphilic molecules and macromolecules, of natural origin or obtained by synthesis from natural raw materials[12].

The list of acronyms used is in the 2.3 section.

This project gathers the competences of research groups with different and complementary methodological and thematic expertises as shown in the table below. International collaborations with Spain, Sweden, Portugal, France and Germany laboratories are active at present.



The lyotropic polymorphism observed in solutions containing surfactants, that originates molecular phases, micelles, precipitates and crystals, vesicles, gels and various liquids crystalline phases varies in a wide concentration range. The addition of polymers modulates the hydrophobic, osmotic, excluded volume, charge density, surface curvature effects of the aggregates. Moreover the added polymers control the dissolving and absorption properties and the viscosity beside giving new technologically remarkable characteristics to these mixtures.

Hydrogels are suited to the controlled release of pharmacologically active molecules[8]. They look extremely promising in this field because they can modulate the released quantity of the drug according to pH, temperature, kind and crosslinking degree. In this context, polymers that respond to small changes of temperature, pH, or to other parameters with discontinuous variation of their volume are defined "intelligent materials" or "stimuli responsive gels" [13]. They have many applications in the biomedical field as materials and systems for the controlled release of drugs.

In the formulation of hydrogels, hydrophobically modified (HMP) hydrosoluble polymers are employed. The interaction of HMP with surfactants have been studied extensively. At polymer low concentrations, the surfactant easily forms a macroscopic and viscoelastic network. The formation of spherical micelles, at higher surfactant concentration, causes a viscosity decrease due to the capture of the polymer hydrophobic groups by the micelles. On the other hand, the interactions with wormlike micelles tend to be cooperative and to increase the viscosity.

Much less is known about the interaction of HMP with vesicles [14]. Such systems are particularly complex when both the HMP and the vesicles bear charge. The HMP-vesicles interactions in ionic solution are modulated by hydrophobic and electrostatic forces at the same time. The polymer chains tend to adsorb on the vesicles, acting as a stabilizing agent. Polymers may cause vesicle fusion or disruptions of the bilayer, too. The addition of an HMP may give rise to vesicle gels by immobilising the vesicles in the polymeric network. Recently only studies concerning synthetic HMP have been published, therefore a study extended also to HMP of natural origin would be important.

The study of systems in which the macromolecule is a protein is quite actual. Many applicative studies concerning the extraction, the precipitation and the denaturation of proteins and the mechanisms of crystallisation in the presence of surfactants have been published. However, some physico-chemical aspects have been almost completely neglected. In particular, aspects of the redissolution mechanism, structure and sizes of the complexes and the peculiar role played by the electrostatic and hydrophobic forces in governing the interaction processes are not known well enough. In order to assess the dependence of the various interactions, above all of the electrostatic ones, between proteins and association colloids on composition, size and charge of the aggregates and/or the protein, it is necessary to deepen the study of the mechanisms of the interaction between proteins and micelles or vesicles, particularly at high concentration (e.g. gels). Vesicles and micelles are two extreme cases, as far as the dimensions of the starting aggregates, their charge density, and therefore the ability of surface adsorption of polymers are concerned. It has not been cleared yet whether the adsorption takes place always with compaction of the biomacromolecules and whether there are saturation effects. In particular, the adsorption of biomacromolecules on the surface of vesicles rules, and then allows, the transfer of the biopolymer in their interior, and the obvious consequences are the transfection technologies, specially of DNA, and the release of biologically active molecules. Therefore it is necessary to study with suited techniques the details connected with the first step of the process, i.e. the surface adsorption, in particular those which cause charge modifications.

Biocompatibility, biodegradation and toxicity of the chemical substances to be used in this research program will be discussed in point 2.3.

Previous scientific activity of the five URs'.

The five UR taking part to the present project have already collaborated in the framework of the program PRIN 2002 having the title "Structure, properties and dynamics of surfactant-macromolecule systems". The most important results obtained are: 75 scientific publications on international journals, 24 graduated in Chemistry and 4 Ph. D. in Chemical Sciences.
The previous results of the groups presenting this project have contributed to gain insight on some aspects relevant to the interactions P-T and to the aggregation of both natural and synthetic surfactants, in sol, gel and liquid crystalline phases.
The instrumental facilities and the experience of the participant UR are essential for the success of this project.

The UR-CS is specialized in advanced NMR methods and Rheology of complex surfactant systems The recent results obtained on the field of the NMR-Diffusometry and NMR-Microscopy, have regarded measurements in aqueous polymer-surfactant mixtures. Polymer, water, surfactant and oil self-diffusion were studied according to classical models as a function of temperature and composition. It was investigated with success the interaction between an ionic surfactant, sodium dodecyl sulphate (SDS), and a self-assembling three-block copolymer, the Pluronic L64 [15,16]. The addition of SDS induced a break-down of anisotropic liquid crystalline phases into isotropic solutions which are bicontinous [17,18].
In recent years, the RU devoted great efforts aimed to perform rheological measurements in concentrated surfactant systems. The rheological properties of lamellar phase of (C10E3)/D2O and (C12E4)/D2O systems have been studied within a wide interval of composition and temperature. A dynamic phase diagram was determined by steady-state rheometry. Under the shear action the lamellar phase was transformed into multilamellar vesicles [19,20]. We reported on the first rheological study of structural relaxations in a nematic liquid crystalline phase. Linear dynamic and transient shear experiments were applied to a poly-domain nematic phase of (CTAB)/water system [21]. The decay of shear modulus in a linear step-strain experiment was analysed using the CONTIN-LT transform and the distribution of relaxation times was calculated.
Novel Rheo-NMR experiments have been recently arranged for interpreting rheological behaviour in microstructural terms. The structural changes of a reverse lecithin-based wormlike phase under steady shear was studied by innovative Rheo-NMR tests [22].

In the framework of the theme of P-T systems in water, the interest of UR-PI has been devoted initially to the classical system PEG-SDS. An extended calorimetric investigation by ITC, coupled with accurate NMR measurements (with UR-TS), allowed to reveal the nature and the stoichiometry of the P-T aggregates as a function of the PEG Mw, put in evidence the stepwise binding of successive micellar clusters on the PEG chain and yielded clear indication of a PEG conformational change.[23] The subsequent thermodynamic study of the more strongly interacting systems PEG-CsPFO [24] and PEG-LiPFN [25], revealed that the relevant aggregates, though characterized by a different stoichiometry and formation energy, exhibit a general behaviour analogous to that exhibited by SDS and also present the same conformational change of the PEG chain. Perfluorinated surfactants form aggregates with PEG more stable and with a higher ionization degree as compared with hydrogenated ones. A further study of the system PEG-LiPFO showed the feeble influence of the nature of the cation on the thermodynamic properties of the P-T aggregates[26].

UR-NA characterized P-T aqueous systems form a physico-chemical and structural point of view by thermodynamic methods (densimetry, differential calorimetry, surface tension), dynamic techniques (mutual and self diffusion), viscosity, spectroscopies (fluorescence, fluorescence quenching, nmr). Recently UR-NA achieved competence in EPR and SANS.
The results have allowed to propose models valuable for the interpretation of the self-organization amphyphilic systems [27,28] and to highlight the influence of the dielectric constant of the solvent [29].
For P-T systems the interaction between several surfactants (alkyl sulfates, sulfonates, ethoxylated) and different polymers: PVP, polyacrilic acid, triblock copolymers, polypeptides, have been studied highlighting some important factors governing the aggregates formation in sol phase such as the hydrophobic, ion-dipole or dipole-dipole interactions [30-35].
Our expertise on mixed micellar systems[36] allowed the formulation and characterization of a surfactant aggregate containing the amide of colecistikinine octapeptide, able to recognize in vivo the tumoral cells [37,38]. The interaction between micelles and a peptide (C-terminal of Gap [39]) put in evidence the ability of micellar-surfactant to trigger aggregation in the peptide itself. This is an important result for the comprehension of the mechanism involved in degenerative patologies such as Alzheimer and BSE.

Recently, the interest towards the NMR of quadrupolar nuclei has enormously increased because innovative experiments can be planned. They are relevant not only to the field of quantum computing [39], but also to the determination of further relaxation parameters, beside T1 and T2, which are connected with structure, order and dynamics in aqueous heterogeneous systems. New computer programs have been developed to obtain such parameters [40].
The UR-TS is active in the field of NMR spectroscopy of quadrupolar nuclei [23,32,41]. Sodium-23 (I= 3/2) is very important for the systems of the present project because it is easy to observe and widely present. The UR-TS had exploited sodium relaxation measurements, jointly with other data obtained by the UR-PI and UR-NA, to study in solution the superaggregation of sodium decyl and dodecyl sulfate with PVP, PEG, PPG, and PEG-PPG triblock polymers (Pluronics) [23,32,43].
In collaboration with UR-RM, it has been studying the aggregation of NaTDC. The BS are peculiar surfactants because they can give rise to quite atypical architectures (helical micelles) even in solution, specially upon addition of NaCl [44]. The signal from the double quantum filtered spectra, also at the magic angle, confirming the presence of large, anisometric aggregates in solutions with high concentrations of NaCl, has been obtained.

Previous UR-RM work on BS dealt with the determination of structural models in crystals, fibers and concentrated BS aqueous solutions to describe the monomer packing in the BS micelles. These models have been verified in the study of micellar aggregates in aqueous solutions of single BS by means of several techniques. XRD, SAXS, DLS, EXAFS, CD, NMR, PGSE-NMR, EPR, EMF, conductometry and dielectric spectroscopy [44-52]. Recently the study of systems formed by two BS has been started (one dihydroxylate +one trihydroxylate or two dihydroxylates), thus providing structural information on mixed micelles [53,54].
In the meantime, SLS and DLS studies on the interaction of DNA with liposome containing Gemini surfactant [55] and on the formation of supramolecular polymers obtained by cyclodextrin host guest complexes [56] have been carried out. In the former, which is particularly interesting in the gene therapy, the influence of the spacer in the surfactant molecule on the DNA compaction has been investigated. Concerning the supramolecular polymers, a crystal structure was also solved thus providing well defined information on the polymer structure [57].
Systematic studies have been performed on the phase diagrams of P-T systems. They have been investigated by thermodynamic, optical, DSC, rheological, 19F NMR. In mixtures containing LYS and LiPFN a rich polymorphic behaviour has been observed, with occurrence of molecular solutions, precipitates, micellar phases, gels and multiphase regions as well. Particularly relevant is the slow nucleation kinetics of the gel phase from the sol and the associated noticeable viscosity increase with time [58]. Similar studies have been performed on the BSA-NaTDC system [59]. There the formation of a gel is observed too; precipitates, if any, occur at pH lower than 5.0.
The physico-chemical properties of hydrophobically modified polysaccharides, whose associative properties depend on the hydrophobic substitution degree, were studied too. The surface coverage kinetics is extremely slow and is controlled by the competition between adsorption and alkyl chain elasticity effects [60]. It has been demonstrated, finally, that non-soluble ionic polyacrylates, obtained by titration with proper counterions and redissolved by SDS in excess, do form P-T complexes. Information from dielectric relaxation experiments put in evidence that saturation occurs for optimal distances between polymer-bound micelles [61]. <<<